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| Status |
Public on May 13, 2021 |
| Title |
A673 STAG2 KO sg1 rep2 |
| Sample type |
SRA |
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| Source name |
A673 Ewing sarcoma cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: A673
genotype/variaton: STAG2 null
crispr-cas9 guides: sgSTAG2-1c6
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNAseq library construction protocol - A673 cells: For the A673 clones, Poly(A) RNA was isolated using the NEBNext mRNA magnetic isolated module (New England Biolabs) and paired-end libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New Englad Biolabs) according to the manufacturer’s protocols with the following modifications: the PCR library enrichment conditions were adjusted to 12 cycles and the PCR library reaction was purified twice by size selection with Agencourt AMPure XP beads (Beckman Coulter). Libraries were subjected to 50 base paired-end sequencing (Illumina HiSeq 2000).
RNAseq library construction protocol - TC71 cells: Poly(A) mRNA was isolated and libraries were prepared using the TruSeq Stranded mRNA Kit (Illumina) according to the manufacturer’s protocol. Strand-specific mRNA seq libraries were pooled and sequenced on a NextSeq500 instrument with single-end 75bp reads to a depth of 30-40M reads/sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
A673.sgSTAG21c6.STAG2null1_R2
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| Data processing |
The CRISPR plasmids pSpCas9(BB)-2A-GFP (PX458) and lentiCRISPR v2 were acquired from Addgene. STAG2 wild-type clones and and STAG2 null clones by CRISPR Cas9 editing were created on A673 cells (STAG2 wild-type: A673.sgNT-1c4, A673.sgNT-2c3, A673.sgLacZ-2c1; STAG2 null: A673.sgSTAG2-1c4, A673.sgSTAG2-1c6, A673.sgSTAG2-4c3, A673.sgSTAG2-4c5) and on TC71 cells (STAG2 wild-type: TC71.sgNT-1c6, TC71.sgNT-2c5; STAG2 null: TC71.sgSTAG2-1c6, TC71.sgSTAG2-4c15, TC71.sgSTAG2-4c18, TC71.sgSTAG2-1c3). Total RNA was extracted with an RNeasy Kit (Qiagen) from STAG2 wild-type and STAG2 null clones (A673.sgNT-1c4, A673.sgNT-2c3, A673.sgLacZ-2c1, A673.sgSTAG2-1c4, A673.sgSTAG2-1c6, A673.sgSTAG2-4c3, A673.sgSTAG2-4c5, TC71.sgNT-1c6, TC71.sgNT-2c5, TC71.sgSTAG2-1c6, TC71.sgSTAG2-4c15, TC71.sgSTAG2-4c18, TC71.sgSTAG2-1c3). For the A673 clones, Poly(A) RNA was isolated using the NEBNext mRNA magnetic isolated module (New England Biolabs) and paired-end libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New Englad Biolabs) according to the manufacturer’s protocols with the following modifications: the PCR library enrichment conditions were adjusted to 12 cycles and the PCR library reaction was purified twice by size selection with Agencourt AMPure XP beads (Beckman Coulter). Libraries were subjected to 50 base paired-end sequencing (Illumina HiSeq 2000). Total RNA was extracted from triplicate samples of TC71 STAG2 clones with the RNeasy Kit and on-column DNA digestion (Qiagen). Poly(A) mRNA was isolated and libraries were prepared using the TruSeq Stranded mRNA Kit (Illumina) according to the manufacturer’s protocol. Strand-specific mRNA seq libraries were pooled and sequenced on a NextSeq500 instrument with single-end 75bp reads to a depth of 30-40M reads/sample.
Quality control tests for the sequenced reads were performed using the FASTQC v0.11.5 software (www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were aligned to the GRCh37/hg19 human genes by using Tophat2 v2.1.1. Quality control tests for the aligned reads and for the replicate consistency were performed by using the qualimap v2.2.1 and the SARTools pipelines.
Gene level reads and gene level expression estimated as log2(1+FPKM) (Fragments Per Kilobase of transcript per Million mapped reads) scores were computed using the Feature Counts method implemented in the Bioconductor v3.5 RSubread v1.5.3 package.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Tab-delimited text file include gene level counts values for each sample.
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| Submission date |
Jul 02, 2018 |
| Last update date |
May 13, 2021 |
| Contact name |
Gabriela Alexe |
| E-mail(s) |
galexe@broadinstitute.org
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| Organization name |
Broad Institute
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| Department |
Computational Biology and Bioinformatics
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| Street address |
415 Main St.
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE116492 |
The effect of STAG2 loss in Ewing sarcoma [RNA-Seq] |
| GSE116495 |
The effect of STAG2 loss in Ewing sarcoma |
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| Relations |
| BioSample |
SAMN09532139 |
| SRA |
SRX4330272 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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